Adenosine plays an important role in the regulation of vascular tone (VT). The effects of adenosine are mediated by G-protein coupled A1, A2A, A2B and A3 adenosine receptors (AR). This laboratory has made a considerable progress towards the understanding on how adenosine interacts with its receptors to regulate VT. Our earlier studies suggest that vascular smooth muscle and endothelium contain more than one AR subtype that may have opposite actions. The studies from this laboratory demonstrated the role of A2A AR (relaxation) and A1 AR (contraction) in the regulation of VT. Also, this laboratory has demonstrated earlier that endothelium may modulate the actions of adenosine through the release of various mediators. Recently, arachidonic acid (AA)-derived metabolites (EETs & 20-HETE) through CYP450s have emerged as important mediators in cardiovascular system. However, the relationship between AR's and these metabolites in the regulation of VT is not well understood. Therefore, our central hypothesis is that the relaxation caused by A2A AR depends on the activity of CYP epoxygenases while the contraction caused by A1 AR depends on the activity of CYP I-hydroxylases. To test this hypothesis, we will use A2A AR-/-, A2A AR+/+, A1 AR-/- and A1 AR+/+ mice aorta employing organ bath; +/- endothelium; AR agonists & antagonists; CYP epoxygenases inhibitors; EETs & 20-HETE agonists & antagonists; measure EETs and 20-HETE formation through UPLC-MS/MS; signaling in aortic smooth muscle & endothelial cells; Western blot and Real-time PCR techniques. We propose four specific aims to determine whether the presence of A2A or A1 AR can have an effect on the VT through: (a) endothelium by preserving the CYP2C activity to generate EETs (EDHF); (b) smooth muscle CYP4A activity to form 20-HETE; (c) PKC and MAPK (ERK1/ERK2) pathways through CYP4A or CYP2C; and finally, (d) opening or blocking ATP-sensitive (KATP) channel through CYP450s (CYP2C & CYP4A). Findings from these studies can lead to better understanding of the signaling pathways for the regulation of VT by ARs involving endothelium, CYP450s, EETs, 20-HETE, PKC, ERK1/ERK2 kinases and KATP channels. The identification of these pathways for AR signaling and their functional significance in the regulation of VT by adenosine may lead to the development of novel pharmacological agents for the treatment of vascular disorders. PUBLIC HEALTH RELEVANCE: Adenosine plays an important role in the maintenance of vascular tone via activation of four receptor (AR) subtypes. In blood vessels, vasodilation is primarily caused by the activation of A2A. Survey of cytochrome 450s (CYP) expression among populations have shown that there is a link between CYP expression and cardiovascular disease, such as hypertension, coronary artery disease, myocardial infarction, heart failure, stroke, cardiomyopathy and arrhythmias. Evidence is mounting for the role of CYP metabolites (EETs or DHETs or 20HETE) in the maintenance of cardiovascular health, including the regulation of vascular tone and cardiac contractility. This proposal is directed towards understanding the relationship between ARs and CYP activity and the relationship between ARs and CYP-generated metabolites (EETs or DHETs or 20HETE). The data generated from this project may lead to better therapeutic approaches involving the imbalance and dysregulation in CYP metabolite(s) formation in cardiovascular diseases.